In vitro Translation Assay with Nsp1

In vitro translation reactions were performed as previously described [30 (link), 31 (link), 32 (link)], with modifications relating to the Nsp1 addition. HEK293F‐cell lysate was pre‐incubated with increasing concentrations (from 0 to 3 μm final concentration) of recombinant Nsp1 (wild‐type and variants) for 15 min on ice. Translation buffer (20 mm HEPES‐KOH, pH 7.6, 1 mm DTT, 0.5 mm spermidine‐HCl, 1 mm Mg(OAc)₂, 8 mm creatine phosphate, 1 mm ATP, 0.2 mm GTP, 150 mm KOAc, 25 μm of each amino acid, and 2 units of human placental ribonuclease inhibitor (Fermentas, Burlington, Canada) was then added followed by 1 μL of the reporter mRNA (0.5 pmol·μL⁻¹) to give a total reaction volume of 13 μL. Final RNA concentration in the reaction mixture was kept at 38 nm. Translation reactions were incubated at 30 °C for 3 h, samples were flash frozen in liquid N₂ and kept at −80 °C. Luciferase assays were performed using the Steady‐Glo Luciferase Assay kit (Promega, Madison, WI, USA) following the manufacturer's protocol. Luminescence was measured using the M200 finite series microplate reader (TECAN, Männedorf, Switzerland). Samples for all concentrations of each protein were prepared in triplicates and measured.